Broth microdilution protocol for determining antimicrobial susceptibility of Legionella pneumophila to clinically relevant antimicrobials

Currently there is no detailed, internationally agreed protocol defined to evaluate antimicrobial susceptibility testing (AST) for Legionella pneumophila (required to establish epidemiological cut-off value or "ECOFF" boundaries); therefore, antimicrobial resistance in these isolates cannot be defined. AST methods utilising media containing activated charcoal as an ingredient, to enable Legionella growth, are unreliable as noted in an internationally authored opinion paper and a new gold standard is required. Here we define a detailed protocol for broth microdilution (BMD) using defined cell culture collection-deposited control reference strains (Philadelphia-1 and Knoxville-1) as well as two accessible reference strains with moderately (lpeAB-carrying) and markedly (23S rRNA mutation-carrying) elevated azithromycin minimum inhibitory concentration (MIC). The defined protocol enables up to eight L. pneumophila strains to be set up on a single 96-well plate per antimicrobial tested. Initial ranges to routinely capture an MIC for these reference strains using clinically relevant antimicrobials azithromycin (0.01-0.25 mg/L), levofloxacin (0.008-0.03 mg/L), lefamulin (0.01-2 mg/L), rifampicin (0.0002-0.0008 mg/L) and doxycycline (0.25-16 mg/L) following incubation for 48 h at 37 °C in a shaking incubator have been empirically determined. Establishment of this internationally agreed protocol sets the scene for the next step: validation and comparison of antimicrobial ranges between international Legionella reference laboratories to establish putative resistance cut-off thresholds for these clinically relevant antimicrobials.